1. Field of the Invention
The present invention relates to the general fields of molecular biology and medical science, and more particularly to integrated microfluidic cartridges for nucleic acid extraction, amplification, and detection from clinical samples.
2. Description of the Related Art
There has been a dramatic transition in clinical laboratory diagnostic assays from the macroscale to the microscale, with specimen volume requirements decreasing from milliliters to microliters, and the possibility of reducing assay times from hours to minutes.
These improvements are due in part to advances in materials and fabrication, to the rapidity of mass and heat transfer at the microscale, and to increases in detection sensitivity, but also represent a continuing effort at innovation.
The engineering of microfluidic devices continues to be the focus of competitive research, and there is a neglected need for improvement in safe handling of fluids. In adapting these devices for clinical diagnosis, special features are needed to guard against and detect false positives, such as from sample contamination, and to protect the operator from exposure to biohazards. Ideally, single-entry devices are needed that seamlessly integrate sample preparation, extraction, and analysis without operator exposure.
PCR (U.S. Pat. Nos. 4,683,195, 4,683,202 and 4,965,188; all incorporated herein by reference) is used to increase the concentration of a target nucleic acid sequence in a sample without cloning, and requires only the availability of target sequence information sufficient to design suitable forward and reverse oligonucleotide primers, typically 10 to 30 base pairs in length. In practice, a molar excess of the primer pair is added to the sample containing the desired target or “template.” The two primers are complementary to 5′ and 3′ sequences of the template respectively. The mixture is first heated to denature or “melt” the double stranded target and then allowed to chill down so as to anneal or “hybridize”, forming mixed primer/target hybrids. Following hybridization, a suitable polymerase can bind to the primer/target hybrids and “extend” the primers along the single stranded template, adding bases at the 3′-OH end of the primer, so as to form a complementary strand. In the presence of both forward and reverse primers, a complete copy of the original double stranded target is made. The steps of denaturation, hybridization, and polymerase extension can be repeated as often as needed to “amplify” the target copy number by several log orders, aiding in its detection. The ultimate number of copies is limited only by the molar quantity of the primers, which—it is important to recognize—are incorporated into the product.
Subsequent to the discovery of PCR, other distinct strategies for amplification were described. See, for example, U.S. Pat. No. 5,130,238 to Malek, entitled “Nucleic Acid Sequence Based Amplification” or NASBA [see also van Gemen et al. 1993. J Virol Methods 43:177-188]; U.S. Pat. No. 5,354,668 to Auerbach, entitled “Isothermal Methodology”; U.S. Pat. No. 5,427,930 to Burkenmeyer, entitled “Ligase Chain Reaction” or LCR [see also European Patent No. 320308; and Schachter et al. 1994. J Clin Microbiol 32:2540-2543]; U.S. Pat. No. 5,455,166 to Walker, entitled “Strand Displacement Amplification” or SDA [see also Walker J et al. 1993. PCR Methods and Applications 3:1-6; Lage J M et al. 2003. Hyperbranched strand displacement amplification. Genome Res 13:294-307; Dean F B 2002. Multiple displacement amplification. Proc NAS 99:5261-66; or Detter J C 2004. Isothermal strand displacement amplification. Genomics 80:691-698], transcription-mediated amplification [see Pfyffer et al. 1996. J Clin Micro 34:834-841]; all of which are incorporated herein by reference. These protocols have various advantages in diagnostic assays, but PCR remains the workhorse in the molecular biology laboratory.
Semi-automated devices for use with the new amplification methodologies followed shortly after the introduction of PCR. The first commercial thermocycler was manufactured under U.S. Pat. No. 5,038,852 to Cetus Corp.
In 1990, the University of Utah (U.S. Pat. No. 6,787,338) disclosed a method wherein samples and reagents were drawn into glass capillaries, which were sealed and placed in an oven, and the temperature was cycled by opening and closing the oven door.
Subsequently, the University of Pennsylvania (U.S. Pat. Nos. 5,498,392; 5,587,128; 5,955,029; 6,953,675) disclosed microfabricated silicon-based devices for performing PCR. Envisaged without particulars was a family of small, mass produced, typically one-use, disposable “chips” for rapid amplification of cellular or microbial nucleic acids in a sample. The devices included a sample inlet port, a “mesoscale” flow system, and a means for controlling temperature in one or more reaction chambers. Heating and cooling means disclosed included electrical resistors, lasers, and cold sinks. Off-chip pumps were used to control fluid flow and to deliver reagents. Printed circuits, sensors on the chip, and pre-analytical binding means for trapping and concentrating analyte were suggested. The common fluid channel, which also served as the analytical channel, was used to transport cell lysis waste (such as bacteria or blood cell lysate) to an open vent or to an off-chip site.
Analytical devices having chambers and flow passages with at least one cross-sectional dimension on the order of 0.1 μm to 500 μm were disclosed. Reaction volumes of 5 μL or lower were prophesized.
Means for detecting amplicons included, nonspecifically, DNA:DNA hybridization, either visually with fluorescent intercalating dyes or through rheological measurement, DNA binding to fluorescent probes or diamagnetic (or paramagnetic) beads; and gel electrophoresis.
While in many ways anticipating current devices, the University of Pennsylvania devices were limited to silicon chips, with sample and reagent ports under the control of external syringe pumps. Cell lysis debris exited the chip through the PCR chamber prior to amplification, and no demonstrable mechanism for isolation of the operator from a biohazardous sample or waste was provided. The design and method did not permit prior on-board incorporation of dehydrated reagents as a single-entry assay device or kit, and notwithstanding any declarations to the contrary, clearly the sharing of pump inlet and outlet ports from sample to sample poses an unacceptable risk for cross-contamination.
In U.S. Pat. No. 5,234,809, a method of purifying nucleic acids is disclosed that involves treating a biological sample, such as blood or stool, with a chaotrope in the presence of a solid substrate such as silicon dioxide or other hydrophilic, cationic solid. Earlier publications had reported the use of chaotropes and solid substrates to purify nucleic acids from agarose blocks. Depending on the nature of the solid phase, the nucleic acid could then be eluted with TE, or not. If not, PCR could be performed directly on the solid substrate, as on nucleic acid trapped on a PVDF membrane. The trapping and eluting step was reported to take about 45 minutes. However, the cited time did not include detection of amplicons. No combination of nucleic acid trapping, amplification and detection of PCR amplicons in a one-step device was disclosed. Interestingly, performance of PCR on eluted filtrates from silica filter pads was not claimed. No multiplexed on-board detection channel was provided.
In U.S. Pat. No. 5,989,813, amplicons are prepared by amplification of target nucleic acid sequences in the presence of forward and reverse primers conjugated with biotin and digoxigenin, respectively, for use in lateral flow assays. The amplicons are bound to particles with streptavidin and agglutinate in the presence of antibody to digoxigenin. By lateral flow, bifunctional amplicon complexes are detected as trapped aggregates excluded from the fibrous matrix. Other solids are interferences in the assay. In a second variant of the lateral flow format, avidin conjugates are wicked into a membrane and migrate until encountering a detection strip. Accumulation of dyed particles at the detection strip is detected. The assays are generally dependent on flow rate in the materials, particle size and pore dimensions as well as laminar barriers to diffusion. No multiplexed on-board detection utility was provided.
Other designs and methods of PCR thermocycling have since been introduced and patented. U.S. Pat. No. 6,210,882 to the Mayo Clinic described means for non-contact heating and cooling for thermocycling reactions. U.S. Pat. No. 5,965,410 to Caliper described means for thermocycling by Joule heating, that is, by the passage of electric current through the buffer of the reaction vessel. U.S. Patent Application 20040081997 to Caliper described PCR reactions in which primers, dNTPs, and the target nucleic acid sequence (template) were first mixed, denatured and re-annealed before polymerase was added (the so-called “hot start” polymerase reaction). Hot Start PCR was earlier suggested to improve product yield and specificity (D'Aquila et al, 1991. Nucleic Acids Res 19:37-49; Chou et al, 1992. Nucleic Acids Res 20:1717-1723; Kellogg et al, 1994. Biotechniques 16: 1134-1137).
Another system for controlling temperature on a microfluidic device is described in U.S. Pat. No. 6,541,274. This patent is directed to a reactor system having a plurality of reservoirs in a body. A heat exchanger and circulating pump is connected with the reservoirs to control the temperature. Other examples of existing devices for controlling temperature on a microfluidic device include radiant heat as described in U.S. Pat. No. 6,018,616, a temperature controlled block as described in U.S. Pat. No. 6,020,187, and other cumulative improvements still being filed with the USPTO.
U.S. Pat. No. 5,716,842 to Biometra described a reactor having a serpentine linear flow microchannel, which crisscrosses heating elements at different temperatures for PCR. U.S. 2001/0046701 to Sentron describes the use of primer attached to particulate reagents for PCR in a serpentine channel followed by absorption-enhanced H-filtration to recover the amplicons. U.S. Pat. No. 5,270,183 describes a reaction chamber coiled around various heating manifolds.
In U.S. Patent Application 2003008308, CalTech described a “rotary microfluidic channel” with multiple temperature zones, so that thermocycling can be performed by circulation of the reaction mixture around an inventive circular channel. The application also teaches the use of accessory channels “formed within an elastomeric material and separated from the flow channel by a section of an elastomeric membrane, the membrane being deflectable into or retractable from the substantially circular flow channel in response to an actuation force applied to the control channel” (Para. 9, FIGS. 3A, 3B). These elastomeric elements were termed “isolation valves” but also served as positive displacement pumps in the devices, again by impinging on the fluid channel under positive pneumatic pressure, whereby the elastomeric element was reversibly deformed and protruded into the fluid channel in the manner of a series of plunger-type peristaltic pump elements. Heating for thermocycling was accomplished with Peltier device, resistive heater, heat exchanger or an indium tin oxide element. By immobilizing the polymerase in one segment of the circular channel not contacted with a hot heating element, use of thermolabile polymerases was suggested.
Proposed detection means included, non-specifically, tagging targets with then-known fluorophores (e.g., as “molecular beacons” or “FRET” tags), chromophores, radioisotopes, luminescence labels, mass labels, enzyme-conjugated oligomeric labels, or by gel electrophoresis. Detection by measuring the capacitance of the reaction solution was disclosed.
U.S. Patent Application 20050019792 to Fluidigm described another elastomeric valve. Other details are provided in U.S. Patent Application 20020195152 to Fluidigm. Pneumatic valves were modified to incorporate a platen which compresses a fluid channel in an elastomeric body, closing or throttling the channel. Devices consisting of blind flow channels, which serve as reaction chambers, and are preloaded with reagents at time of manufacture, were also proposed.
U.S. Patent Application 20060073484 to Mathies described single channel or dense network microfluidic devices under control of a pneumatic manifold. The method involved the use of immunoaffinity capture of target pathogens, followed by lysis and detection by polymerase chain reaction (PCR) with capillary electrophoresis (CE). Pneumatically switchable control valves, consisting of a PDMS elastic film sandwiched between a glass fluidic and pneumatic manifold layer (with optional via layer interposed), were used to either open channels by applying vacuum or close channels by applying pressure (see FIG. 1C of U.S. Patent Application 20060073484). Similar structures were also used as pumps for dispensing reagents (see FIG. 5C of U.S. Patent Application 20060073484). [See also, William H. Grovera et al. 2003. Monolithic membrane valves and diaphragm pumps for practical large-scale integration into glass microfluidic devices. Sensors and Actuators B 89(3):315-323.] PDMS films were particularly preferred because silane elastomers bond to the glass plates of these devices.
U.S. Patent Application 20050129582 co-assigned to Micronics teaches microfluidic devices having channels, valves, pumps, flow sensors, mixing chambers and optical detectors for performance of chemical and biochemical assays, and is herein incorporated in full by reference. Improved thermal transitions were disclosed, enabling performance of PCR more than 4 times faster than conventional thermocyclers. Thermal ramping rates of up to 17 C/sec were demonstrated by selection of suitable plastic substrates and dimensions.
Co-assigned patents and patent applications relevant to the development methods for nucleic acid and antibody bioassays in a microfluidic assay format include U.S. Pat. No. 6,743,399 (“Pumpless Microfluidics”), U.S. Pat. No. 6,488,896 (“Microfluidic Analysis Cartridge”), U.S. Patent Applications 2005/0106066 (“Microfluidic Devices for Fluid Manipulation and Analysis”), 2002/0160518 (“Microfluidic Sedimentation”), 2003/0124619 (“Microscale Diffusion Immunoassay”), 2003/0175990 (“Microfluidic Channel Network Device”), 2005/0013732 (“Method and system for Microfluidic Manipulation, Amplification and Analysis of Fluids, For example, Bacteria Assays and Antiglobulin Testing”), U.S. Patent Application 2007/0042427, “Microfluidic Laminar Flow Detection Strip”, and unpublished documents “Microfluidic Cell Capture and Mixing Circuit”, “Polymer Compositions and Hydrogels”, “Microfluidic Mixing and Analytical Apparatus,” “System and method for diagnosis of infectious diseases”, and “Microscale Diffusion Immunoassay Utilizing Multivalent Reactants”, all of which are hereby incorporated in full by reference.
Other illustrations of microfluidic devices and their components may be found in U.S. Pat. Nos. 5,726,751; 5,724,404; 5,716,852; 5,747,349; 5,748,827; 5,922,210; 5,932,100; 5,974,867; 5,971,158; 5,972,710; 5,948,684; 6,007,775; 6,171,865, and 6,387,290 (hereby incorporated by reference in their entirety).
U.S. Pat. No. 5,582,989 teaches the use of multiplex PCR to detect genetic errors in multiple target exons. Purified gDNA was prepared by the standard methods of the time, followed by PCR, with detection by agarose gel electrophoresis, or by Southern blot. A simplified, integrated microfluidic approach to genetic analysis was not anticipated.
U.S. Pat. No. 5,582,989, incorporated herein by reference, teaches the use of multiplex PCR to detect infectious agents in clinical samples. The key teaching of the procedure is a high-salt buffer used to extract the DNA from proteins, which are salted out. DNA was then further purified by phenol:chloroform extraction before PCR. Finally, PCR was performed using a Perkin Elmer 9600 thermocycler (Norwalk, Conn., USA). The protocol was described as taking about a day to complete. No on-board multiplexed detection utility was provided.
U.S. Pat. No. 7,087,414 to Applera described a two stage assay including a preparative step in which a mixed primer pool was used for preliminary rounds of amplification (to no more than 1000 copies per target), and the reaction mixture was then split into separate parallel reaction microwells for subsequent second-stage amplifications, termed “boost” cycles, in which single, target-specific primer pairs were used. Amplification was followed by detection in separate analytical channels. Microfluidic devices with integrated one-step extraction, amplification and detection were not anticipated. No on-board multiplexed detection utility was provided.
Accordingly, there remains an unfulfilled need for an integrated, one-step microfluidic assay device capable of sample processing and nucleic acid capture, amplification of target sequences, and detection of positive results. There is a need for devices that deliver results in real time with minimal delay. This need is particularly apparent in the fight against infectious diseases—where patients must be evaluated and treated without the option of a return visit—where rapid identification of an etiological agent is used for real time monitoring and control of epidemics—where specialized facilities for handling biohazardous samples may not be available—where the testing must be performed absent skilled microbiologists—and where the cost of more complex diagnostic services is prohibitive.
Ease of use would be improved if multiple results were displayed in parallel, as for example the results of a panel of tests, in a user friendly, visual detection chamber.